Mayumi Furuya

C-type natriuretic peptide is a growth inhibitor of rat vascular smooth muscle cells.

C-type natriuretic peptide (CNP) which potently stimulates particulate guanylate cyclase activity in cultured rat vascular smooth muscle cells (VSMC) inhibited serum-induced DNA synthesis of the cells 10-fold more effectively than alpha-human atrial natriuretic peptide (alpha-hANP). The inhibitory effect of CNP was mimicked by 8-bromo-cGMP. The proliferation of VSMC was also suppressed by CNP more potently than alpha-hANP, while the peptide was less active for cGMP augmentation and for vasorelaxation than alpha-hANP in isolated rat aorta. These results suggest that CNP may be a growth regulating factor of VSMC rather than a vasodilator.

Local Expression of C-Type Natriuretic Peptide Markedly Suppresses Neointimal Formation in Rat Injured Arteries Through an Autocrine/Paracrine Loop

BACKGROUND In vivo gene transfer into injured arteries may provide a new means to facilitate molecular understanding of and to treat the intractable fibroproliferative arterial diseases. Selection of an optimal molecule to be transferred will be a key to successful gene therapy in the future. We tested the hypothesis that a secreted multifactorial molecule should act more efficiently through an autocrine/paracrine loop to suppress neointimal formation elicited in injured arteries than a simple growth-inhibiting molecule that might be expressed inside cells. METHODS AND RESULTS We constructed an adenoviral vector (AdCACNP) expressing C-type natriuretic peptide (CNP), a secreted stimulator of membrane-bound guanyl cyclase. AdCACNP directs cells to secrete large quantities of biologically active CNP. Serum-stimulated DNA synthesis and cell proliferation were only moderately suppressed in arterial smooth muscle cells infected with AdCACNP in vitro. However, when AdCACNP was applied to balloon-injured rat carotid arteries in vivo, neointimal formation was markedly reduced (90% reduction) in an infection-site-specific manner without an increase in plasma CNP level. CONCLUSIONS Our results showed that CNP, a secreted multifactorial molecule, was indeed effective in suppressing fibroproliferative response in injured arteries and suggest that the potent antiproliferation effect may not be the most critical factor for the effective suppression of neointimal formation. An adenovirus-mediated expression of CNP could be an effective and site-specific form of molecular intervention in proliferative arterial diseases.

Novel natriuretic peptide, CNP, potently stimulates cyclic GMP production in rat cultured vascular smooth muscle cells.

The newly identified peptide C-type natriuretic peptide (CNP) caused only a slight elevation of cGMP in rat renal glomeruli. In contrast, CNP potently increased cGMP levels in cultured rat vascular smooth muscle cells (VSMC) and stimulated guanylate cyclase activity in the particulate fraction of the cells. The extent of maximum activation of the enzyme induced by CNP was 4-fold higher than that by human atrial natriuretic peptide (alpha-hANP) while CNP was 4- and 16-fold weaker than alpha-hANP in binding affinity for the putative receptors on VSMC and vasorelaxant activity for rat aorta, respectively. These results indicate that CNP is a potent stimulator of cGMP formation in VSMC but not in glomeruli and pharmacological feature of CNP is distinct from that of ANP.

Structural requirements of C-type natriuretic peptide for elevation of cyclic GMP in cultured vascular smooth muscle cells

C-type natriuretic peptide (CNP), which was recently found to be a selective ligand for one of the two known natriuretic peptide receptor guanylyl cyclases (NPR-B), potently stimulates cGMP production in cultured rat vascular smooth muscle cells (VSMC) and exerts potent antiproliferative effects on the cells. To investigate the structural requirements of CNP for stimulation of cGMP accumulation via NPR-B, we prepared CNP analogs and tested them on cultured rat VSMC. Our results indicate that only the ring portion of CNP with a disulfide bond (CNP(6-22)) participates in stimulation of cGMP accumulation, especially the sequence Leu9-Lys10-Leu11 in the ring portion executes essential roles for both elevation of cGMP and selectivity of the ligand for NPR-B. We also found a good correlation between the activities of the CNP analogs for stimulation of cGMP accumulation and inhibition of DNA synthesis.

Ghrelin improves body weight loss and skeletal muscle catabolism associated with angiotensin II-induced cachexia in mice

Ghrelin is a gastric peptide that regulates energy homeostasis. Angiotensin II (Ang II) is known to induce body weight loss and skeletal muscle catabolism through the ubiquitin-proteasome pathway. In this study, we investigated the effects of ghrelin on body weight and muscle catabolism in mice treated with Ang II. The continuous subcutaneous administration of Ang II to mice for 6 days resulted in cardiac hypertrophy and significant decreases in body weight gain, food intake, food efficiency, lean mass, and fat mass. In the gastrocnemius muscles of Ang II-treated mice, the levels of insulin-like growth factor 1 (IGF-1) were decreased, and the levels of mRNA expression of catabolic factors were increased. Although the repeated subcutaneous injections of ghrelin (1.0mg/kg, twice daily for 5 days) did not affect cardiac hypertrophy, they resulted in significant body weight gains and improved food efficiencies and tended to increase both lean and fat mass in Ang II-treated mice. Ghrelin also ameliorated the decreased IGF-1 levels and the increased mRNA expression levels of catabolic factors in the skeletal muscle. IGF-1 mRNA levels in the skeletal muscle significantly decreased 24h after Ang II infusion, and this was reversed by two subcutaneous injections of ghrelin. In C2C12-derived myocytes, the dexamethasone-induced mRNA expression of atrogin-1 was decreased by IGF-1 but not by ghrelin. In conclusion, we demonstrated that ghrelin improved body weight loss and skeletal muscle catabolism in mice treated with Ang II, possibly through the early restoration of IGF-1 mRNA in the skeletal muscle and the amelioration of nutritional status.

Possible involvement of melanocortin-4-receptor and AMP-activated protein kinase in the interaction of glucagon-like peptide-1 and leptin on feeding in rats

Glucagon-like peptide-1 (GLP-1) and leptin are anorectic hormones produced in the small intestine and white adipose tissue, respectively. Investigating how these hormones act together as an integrated anorectic signal is important to elucidate a mechanism to maintain energy balance. In the present study, coadministration of subthreshold GLP-1 and leptin dramatically reduced feeding in rats. Although coadministration of GLP-1 with leptin did not enhance leptin signal transduction in the hypothalamus, it significantly decreased phosphorylation of AMP-activated protein kinase (AMPK). In addition, coadministration of GLP-1 with leptin significantly increased proopiomelanocortin (POMC) mRNA levels. Considering that α-melanocortin stimulating hormone (α-MSH) is derived from POMC and functions through the melanocortin-4-receptor (MC4-R) as a key molecule involved in feeding reduction, the interaction of GLP-1 and leptin on feeding reduction may be mediated through the α-MSH/MC4-R system. As expected, the interaction of GLP-1 and leptin was abolished by intracerebroventricular preadministration of the MC4-R antagonists agouti-related peptide and SHU9119. Taken together, GLP-1 and leptin cooperatively reduce feeding at least in part via inhibition of AMPK following binding of α-MSH to MC4-R.

Discovery of a non-peptide small molecule that selectively mimics the biological actions of calcitonin.

Calcitonin (CT), a 32-amino acid peptide hormone secreted mainly from the thyroid gland, plays an important role in maintaining bone homeostasis. To discover non-peptide small molecules with biological actions similar to those of CT, a cell-based screening of an in-house chemical library was performed and a pyridone derivative (SUN B8155) was identified. Like CT, it elevated cyclic AMP (cAMP) levels in T47D and UMR106-06 cells which endogenously express human and rat CT receptor, respectively. SUN B8155 also stimulated cAMP formation in cells expressing recombinant human CT receptor, but not in those expressing human parathyroid hormone/parathyroid hormone-related peptide receptor. Accumulation of cAMP in T47D cells was blocked by a selective antagonist of CT receptor, salmon CT(8-32), whereas SUN B8155 did not displace the specific binding of [(125)I]CT to the receptor. Our results suggested that the compound selectively interacts with the CT receptor by a mechanism similar to but probably different from that of CT itself. In rats, intraperitoneal administration of SUN B8155 significantly lowered serum calcium levels, like CT. Our results demonstrate, for the first time, that the biological activities of the newly identified small molecule can mimic that of CT, acting via the CT receptor.

The role of C-terminal part of ghrelin in pharmacokinetic profile and biological activity in rats.

Ghrelin is an endogenous ligand for growth hormone secretagogue receptor 1a (GHS-R1a), and consists of 28 amino acid residues with octanoyl modification at Ser(3). The previous studies have revealed that N-terminal part of ghrelin including modified Ser(3) is the active core for the activation of GHS-R1a. On the other hand, the role of C-terminal (8-28) region in ghrelin has not been clarified yet. In the present study, we prepared human ghrelin, C-terminal truncated ghrelin derivatives and anamorelin, a small molecular GHS compound which supposedly mimics the N-terminal active core, and examined GHS-R1a agonist activity in vitro, pharmacokinetic (PK) profile and growth hormone (GH) releasing activity in rats. All compounds demonstrated potent GHS-R1a agonist activities in vitro. Although the lack of C-terminal two amino acids did not modify PK profile and GH releasing activity, the deletion of C-terminal 8 and 20 amino acids affected them, and ghrelin(1-7)-Lys-NH(2) exhibited very short plasma half-life and low GH releasing activity in vivo. In rat plasma, ghrelin(1-7)-Lys-NH(2) was degraded more rapidly than ghrelin, suggesting that C-terminal part of ghrelin protected octanoylation of Ser(3) from plasma esterases. Subdiaphragmatic vagotomy significantly attenuated GH response to ghrelin but not to anamorelin. These results suggest that the C-terminal part of ghrelin has an important role in the biological activity in vivo. We also found that ghrelin stimulated GH release mainly via a vagal nerve pathway but anamorelin augmented GH release possibly by directly acting on brain in rats.

Rapid and Mild Generation of Carbon Radicals fromo-(o-Iodophenyl)phenylthio Derivatives by an Anchimeric Approach

Keywords: carbon radical generation; cyclization; o-(o-iodophenyl)phenylthio derivative; nucleophilic thiolation; reduction

Metabolic features of rats resistant to a high-fat diet.

OBJECTIVE We assessed the metabolic characteristics of high-fat-diet-resistant (DR) rats. METHODS Body weight, energy intake, locomotor activity, oxygen consumption, plasma leptin and lipid levels, size of visceral-fat adipocytes, and mRNA levels of genes related to lipid metabolism were measured in control rats fed standard chow and in obesity-prone (high-fat-diet-induced obesity, DIO) and DR rats fed a high-fat diet. Glucose tolerance and insulin tolerance tests were also performed. RESULTS DIO rats gained weight more rapidly than did DR and control rats; DR rats gained less weight than did DIO rats despite similar energy intake. Energy expenditure did not differ among the three groups. The diameter of visceral-fat adipocytes was similar in DR and control rats. mRNA levels of genes involved in lipogenesis, such as fatty acid synthase and acetyl-CoA carboxylase, tended to be lower in DR than in control and DIO rats, whereas those of carnitine palmitoyltransferase 1a, which is involved in fatty acid β-oxidation, were greater in DR rats than in the other groups. DIO rats showed hyperinsulinemia and glucose intolerance, whereas DR rats had high sensitivity to insulin. CONCLUSION DR rats show suppression of lipogenesis and acceleration of fatty acid β-oxidation in the visceral fat. These characteristics likely contribute to the anti-obesity phenotype of DR rats.